Universal capture array for multiplexed subtype-specific quantification and stability determination of influenza proteins

Abstract

A universal array for multiplexed quantification of variable hemagglutinin such as subcomponents of multivalent annual influenza vaccines that is robust to variations in proteins such as mutations and is capable of quantifying degradation of proteins. Universal capture array ( 100 ) comprises one or more substrates ( 102 ) and a low-density microarray ( 104 ) of sub-arrays ( 108 ) comprising spots ( 106 a - c ). The microarray ( 104 ) is contacted with one or more targets ( 202 ) at one or more unknown concentrations, and bound complexes ( 203 ) are formed and subsequently quantified with a suitable method. Quantified signals are compared to calibration curves to obtain one or more unknown concentrations and/or quantify degradation of the one or more targets ( 202 ). Other embodiments are described and shown.

What is claimed is: 
     
       1. A method for determining one or more unknown concentrations of hemagglutinin proteins in a vaccine sample, the method comprising:
 providing one or more substrates comprising at least one microarray, each microarray comprising:
 i. at least one positive control spot, 
 ii. at least one negative control spot, and 
 iii. more than one subarray, each subarray including a plurality of capture agent spots, each capture agent spot including a murine antibody configured to bind to a linear or conformational epitope of an influenza hemagglutinin protein, wherein a concentration of the antibody in each of the plurality of capture agent spots is the same; 
 
 contacting at least one microarray with the sample to form one or more bound complexes, each of the bound complexes comprising a capture agent and an influenza hemagglutinin protein; 
 generating one or more signals corresponding to an amount of the one or more bound complexes at each spot; 
 quantifying the one or more signals to generate one or more quantified signals; 
 providing one or more calibration curves that establish a one-to-one relationship between a concentration of a reference hemagglutinin protein and the quantified signals; 
 comparing the one or more quantified signals to the one or more calibration curves to determine the one or more unknown concentrations of the hemagglutinin proteins in the sample; and 
 the microarray comprises a plurality of capture agents that are different but related capture agents, wherein a capture agent targets one subset of a class of an antigen in the sample and a different capture agent targets a different subset of the same class of the antigen, wherein the microarray further comprises:
 a first subarray having capture agent spots comprising a first anti-A/H1 monoclonal antibody to bind to a first conformational epitope of influenza hemagglutinin; 
 a second sub-array having capture agent spots comprising a second anti-A/H1 monoclonal antibody to bind to a second conformational epitope of influenza hemagglutinin; 
 a third sub-array having capture agent spots comprising a first anti-A/H3 monoclonal antibody to bind to a third conformational epitope of influenza hemagglutinin; 
 a fourth sub-array having capture agent spots comprising a second anti-A/H3 antibody to bind to a fourth conformational epitope of influenza hemagglutinin; 
 a fifth sub-array having capture agent spots comprising a first anti-B/Yamagata monoclonal antibody to bind to a fifth conformational epitope of influenza hemagglutinin; 
 a sixth sub-array having capture agent spots comprising a second anti-B/Yamagata monoclonal antibody to bind to a sixth conformational epitope of influenza hemagglutinin; 
 a seventh sub-array having capture agent spots comprising a first anti-B/Victoria monoclonal antibody to bind to a seventh conformational epitope of influenza hemagglutinin; and 
 an eighth sub-array having capture agent spots comprising a second anti-B/Victoria monoclonal antibody to bind to an eighth conformational epitope of influenza hemagglutinin, 
 
 wherein the sub-arrays collectively enable the microarray to detect hemagglutinin proteins associated with an influenza of at least one of A/H1, A/H3, B/Yamagata HA or B/Victoria antigen over a period selected from the range of one to six years despite antigenic drift, thereby eliminating a need to change the plurality of capture agents to adapt to seasonal influenza mutations. 
 
     
     
       2. The method of  claim 1 , wherein at least one of the sub-array groups targets at least one possible mutation of influenza A hemagglutinin subtype 1. 
     
     
       3. The method of  claim 1 , wherein at least one of the sub-array groups targets at least one possible mutation of influenza A hemagglutinin subtype 3. 
     
     
       4. The method of  claim 1 , wherein at least one of the sub-array groups targets at least one possible mutation of Yamagata-lineage influenza B hemagglutinin. 
     
     
       5. The method of  claim 1 , wherein at least one of the sub-array groups targets at least one possible mutation of Victoria-lineage influenza B hemagglutinin. 
     
     
       6. The method of  claim 1 , wherein the microarray comprises at least 4 sub-array groups, one of the at least 4 sub-array groups targeting influenza A hemagglutinin subtype 1, one of the at least 4 sub-array groups targeting influenza A hemagglutinin subtype 3, one of the at least 4 sub-array groups targeting Yamagata-lineage influenza B hemagglutinin, and one of the at least 4 sub-array groups targeting Victoria-lineage influenza B hemagglutinin. 
     
     
       7. The method of  claim 1 , wherein the one or more signals are fluorescence intensities. 
     
     
       8. The method of  claim 1 , wherein the one or more signals are quantified using a microarray scanner. 
     
     
       9. The method of  claim 1 , wherein quantifying the one or more signals includes normalizing the signals as a function of a signal associated with the positive control spot and/or as a function of a signal associated with the negative control spot. 
     
     
       10. The method of  claim 1 , wherein the substrate comprises at least 4 microarrays. 
     
     
       11. The method of  claim 10 , wherein the calibration curves are provided by at least one microarray of the substrate. 
     
     
       12. The method of  claim 1 , wherein the microarrays each include at least 4 subarrays. 
     
     
       13. The method of  claim 1 , wherein the microarrays comprise:
 at least one antibody capture agent capable of binding to a hemagglutinin associated with a first influenza subtype or lineage, and 
 at least one antibody capture agent capable of binding to a hemagglutinin associated with a second influenza subtype or lineage, and 
 wherein the step of comparing simultaneously determines a concentration of the hemagglutinin associated with the first influenza subtype or lineage and a concentration of the hemagglutinin associated with the second influenza subtype or lineage. 
 
     
     
       14. The method of  claim 1 , wherein the microarrays comprise:
 a combination of antibody capture agents specific to a predetermined plurality of influenza subtypes and/or lineages, and 
 wherein the step of generating comprises contacting the bound complexes with a single label agent capable of binding to hemagglutinin associated with each of the predetermined influenza subtypes and/or lineages, and 
 wherein the step of comparing simultaneously determines a concentration of the hemagglutinin proteins from each of the predetermined influenza subtypes and/or lineages. 
 
     
     
       15. The method of  claim 1 , wherein the microarrays comprise:
 at least one antibody capture agent capable of binding to a conformational epitope of a hemagglutinin protein associated with each of a plurality of predetermined influenza subtypes and/or lineages, 
 and at least one antibody capture agent capable of binding to a linear epitope of the hemagglutinin protein associated with each of the plurality of predetermined influenza subtypes and/or lineages, and 
 wherein the step of comparing simultaneously determines a concentration of the hemagglutinin proteins from each of the predetermined influenza subtypes and/or lineages and further simultaneously determines a stability of the hemagglutinin proteins from each of the predetermined influenza subtypes and/or lineages. 
 
     
     
       16. The method of  claim 1 , wherein the microarrays comprise:
 at least one neutralizing antibody capture agent capable of binding to a hemagglutinin protein associated with each of a plurality of predetermined influenza subtypes and/or lineages, and 
 at least one non-neutralizing antibody capture agent capable of binding to a hemagglutinin protein associated with each of the plurality of predetermined influenza subtypes and/or lineages, and 
 wherein the step of comparing simultaneously determines a concentration of the hemagglutinin proteins from each of the predetermined influenza subtypes. 
 
     
     
       17. The method of  claim 1 , wherein the microarrays comprise:
 at least one neutralizing antibody capture agent capable of binding to a conformational and linear epitope of a variable HA1 region of a hemagglutinin protein associated with an influenza strain, and 
 at least one neutralizing antibody capture agent capable of binding to a conformational and linear epitope of a conserved HA2 region of a hemagglutinin protein associated with the influenza strain, and 
 wherein the step of comparing determines a stability of the hemagglutinin protein. 
 
     
     
       18. The method of  17 , wherein the step of generating comprises contacting the bound complexes with a single label agent capable of binding to hemagglutinin associated with each of the predetermined influenza subtypes and/or lineages, and wherein the step of comparing simultaneously determines a concentration and a stability of the hemagglutinin protein. 
     
     
       19. The method of  17 , wherein the step of generating comprises contacting the bound complexes with a single label agent capable of binding to hemagglutinin associated with each of the predetermined influenza subtypes and/or lineages, and
 wherein the step of comparing simultaneously determines a concentration of the hemagglutinin proteins from each of the predetermined influenza subtypes and/or lineages. 
 
     
     
       20. The method of  claim 1 , wherein the substrate further includes a mask surrounding the microarrays. 
     
     
       21. The method of  claim 1 , wherein the vaccine sample is a multivalent annual influenza vaccine comprising antigens from the influenza subtypes A/H1 and A/H3, and lineages B/Yamagata HA and B/Victoria, and the method simultaneously differentiates and quantifies antigens associated with each of the influenza subtypes A/H1 and A/H3, and lineages B/Yamagata HA and B/Victoria. 
     
     
       22. The method of  claim 1 , wherein the vaccine sample is a monovalent formulation for use as a component in a multivalent influenza vaccine. 
     
     
       23. The method of  claim 1 , wherein the microarray further comprises:
 capture agent spots comprising a third anti-A/H1 antibody to bind to a first linear epitope of influenza hemagglutinin; and/or 
 capture agent spots comprising a third anti-A/H3 antibody to bind to a second linear epitope of influenza hemagglutinin.